Platelets contain a unique chemokine Platelet Factor 4 (PF4), which is released in large amounts at sites of platelet activation. While a number of potential roles of PF4 in the cardiovascular system have been suggested based on in vitro data, its in vivo biological role(s) is unclear. In this application we will define the role of PF4 in platelet biology, extending our preliminary in vitro data of PF4's interaction with low-density lipoprotein (LDL), with its receptor LDL-R and with the related receptor LRP. We will also extend these observations in vivo. Specific aims include: Number 1: Characterize PF4's interactions with LDL-R and LRP in vitro. These studies will characterize the interactions of PF4 with LDL binding to the LDL-receptor (R), identifying the responsible domains within PF4 and LDL-R using defined chimeras and mutants, and exploring the hypothesis that oligomerization of PF4 by cellular proteoglycans frustrates the endocytosis of LDL/LDL-R complexes. We will also examine the specificity of PF4's interactions with LRP and further characterize its ability to influence the uptake of urokinase into cells. Number 2: Effect of PF4 on the development of atherosclerosis focusing on a transgenic mice approach. We have created PF4 null mice (mPF4) and mice overexpressing human PF4 (hPF4+). These animals will be studied to see the effect of PF4 level on LDL metabolism. The animals will also be crossed onto several well-defined atherogenic models that will test specific issues related to PF4's affect on LDL metabolism and the development of atherosclerosis. Number 3: Effect of PF4 on the cardiovascular biology focusing on a transgenic mice approach. Strong in vitro data developed by our group and others have suggested a role of PF4 in the development of thrombosis, angiogenesis and megakaryopoiesis. However, a physiologic role of PF4 in these processes has yet to be demonstrated. In this specific aim, we plan to use the mPF4-/- and hPF4+ mice to begin to define the biological relevance of the in vitro studies in the literature and presented in the 1st specific aim. Crosses onto other transgenic animal models that will enhance our understanding of the molecular mechanism(s) by which PF4 contributes to these biological processes are also proposed. We believe that new information derived by this application will have clinical relevance. The biological studies of PF4 should provide new insights into the regulation of several cardiovascular-related processes, and may offer novel approaches for the treatment of cardiovascular diseases.